Crawling cells in the body must traverse a complex landscape obstructed by cell-cell junctions, extracellular matrix, and other cells. The elegant and apparently effortless navigation of neutrophils through tissue in tension at one moment and in compression the next raises questions about how motile cells respond to external as well as self-imposed loads. More specifically, how are the actin filament networks that generate cellular protrusions during motility influenced by the forces on them? The common view of dynamic actin networks as having properties governed only by concentrations of actin and binding proteins is not sufficient to explain how cells adapt to their physical environment;the role of forces in restructuring or remodeling actin networks must be considered. We hypothesize that external loads mechanically regulate actin network properties by altering network architecture, such as filament density and crosslinking, in response to changes in loading conditions. This `tuning'or adaptation of actin network mechanical properties and growth rates in response to load may be an important mechanism for guiding crawling cells and play critical roles in other actin-based processes including endocytosis, phagocytosis, and mechanotransduction. However, basic questions about the physical behavior of dynamic actin networks remain unanswered. This proposal focuses on the response of Arp2/3-branched actin networks to forces. Using an in vitro model of actin network growth, we ask several basic questions about mechanical properties, growth rates, and the role of actin binding proteins in organizing network architecture. We have developed a dual-cantilever atomic force microscope (AFM) with time-lapse fluorescence microscopy that has the unique ability to measure both static and dynamic actin network properties under controlled loading conditions. In preliminary experiments using this technique, we have identified complex mechanical behavior of actin networks under high loads and growth rates that depend on loading history rather than instantaneous load. This proposal will use a minimal system of purified proteins that form growing actin networks to (i) identify the effect of loading on mechanical properties, (ii) track changes in growth rates in response to force, and (iii) examine the transient effect of an actin filament severing protein on network properties. The proposed work will impact health by revealing actin network mechanics that play a fundamental role in cellular protrusion essential for cell movements during development as well as metastasis. PUBLIC HEALTH RELEVANCE Dynamic actin networks have been identified as essential for not only cell motility, but also endocytosis, exocytosis, pathogen invasion, T-cell signaling, invadopodia formation, and cell division. Better understanding of how the mechanical microenvironment alters actin network behavior, including its elastic properties and its ability to generate and direct forces for shape change, has the potential to reveal new mechanisms of disease and identify new targets for drug discovery.